Aflatoxins, mycotoxins of fungi of the Aspergillus sp., pose a risk to consumer health and are, therefore, regulated by more than 100 countries. To facilitate method development and validation as well as assessment of measurement capabilities, availability of certified reference materials and proficiency testing schemes is important. For these purposes, highly accurate determinations of the aflatoxin content in the materials used are necessary. We describe here the use of two-dimensional heart-cut LC-LC in combination with exact-matching double isotope dilution mass spectrometry to determine the content of aflatoxin B1 in three materials used in a proficiency testing scheme. The serious reduction in ionization suppression afforded by the two-dimensional heart-cut LC-LC had a positive effect on the precision of the measured isotope ratios of the exact-matching double isotope dilution mass spectrometry. This is evidenced by the expanded measurement uncertainty (k=2) of 0.017 μg/kg or 8.9 % relative to a mass fraction of aflatoxin B1 in a cereal-based baby food of 0.197 μg/kg. This value is in perfect agreement with the consensus value of this material from a proficiency test (PT) scheme for National Reference Laboratories executed by the European Reference Laboratory for Mycotoxins. The effort necessary to perform the described methodology precludes its frequent use but for specific applications we see it as a valuable tool.